rabbit anti-integrin α 3β 1 Search Results


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  • 99
    Millipore anti α tubulin
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    Cell Signaling Technology Inc akt
    Overview of the mechanism by which MMP-9 regulates formation of focal adhesion junctions. (A) During exocytosis and upon being secreted into the extracellular space, proMMP-9 forms a large complex with itself that can then act as a scaffold for promoting outside-in signaling. In our model, PEX-9 scaffolding promotes the association of <t>β1</t> integrin-EGFR-CD44. Interaction with CD44 results in enhanced EGFR activation in addition to increased phosphorylation of its downstream targets <t>AKT</t> and Erk 1 + 2 (through the MAPK/Erk pathway). While complexed to β1 integrin, EGFR then goes on to transactivate Src kinase after it is recruited to the α4 integrin subunit during the generation of a new focal adhesion contact site. After activation, Src can then directly phosphorylate FAK currently associated with the β1 integrin subunit resulting in its maximal catalytic activity. This active Src–FAK complex can then bind and activate PAX, resulting in a mature FAK-PAX complex necessary for the formation of focal adhesion junctions. Formation of a complex between FAK and PAX results in final translocation to ECM–integrin junctions at the cell surface where they regulate cytoskeletal interactions resulting in enhanced cellular adhesion, migration, and invasion of cancer cells. (B) Treatment with PEX-9 inhibitor (depicted as red triangle) prevents MMP-9 scaffolding, thereby preventing downstream signaling driven by EGFR activation.
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    Cell Signaling Technology Inc β catenin
    E2F1 abrogates Wnt signaling by modulating <t>β-catenin</t> target gene expression and inducing the GSK3-independent degradation of β-catenin ( a ) In Saos2- TR-E2F1 cells, E2F1 represses c-myc levels without affecting the levels of TCF1 or TCF4. ( b–d ) E2F1 modulates the expression of endogenous Wnt target genes (qPCR analysis after Tet-induced E2F1 expression at 24 hours). ( e ) The kinetics of E2F1-induced AXIN2 and SIAH1 expression mirrors the E2F1 activation of CCNE1/Cyclin E and CCNA1/Cyclin A , and precedes E2F1-induced apoptosis. ( f ) E2F1 represses the expression of Wnt targets in DLD1 colorectal cancer cells. Levels of mRNA were normalized to GAPDH and the effect of E2F1 is depicted as the ratio between samples after pCMV-empty or pCMV-E2F1 (1 µg each) expression. ( g, h ) is both GSK3- and caspase-independent. Saos2 cells were treated with control (DMSO), GSK3 inhibitors (20 µM SB216763 and 5 mM LiCl), or the caspase inhibitor peptide BOC-aspartyl-FMK (BAF; 100 µM) with or without Tet-induction of E2F1 (Western blot). ( i ) Co-expression of Bcl-2 (25 ng), pRb (10–25 ng), stabilized tumor-derived β-catenin mutants (10–25 ng), or the GSK3β inhibitor SB216763 (15 uM), is sufficient to partially-rescue E2F1-induced apoptosis. Cell death was depicted as percent inhibition of EGFP loss at 48 hours after transfection with pCMV-E2F1 or pCMV-empty (100 ng each) along with EGFP expression construct. All data is expressed as mean ± s.d. (n = 3).
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    Cell Signaling Technology Inc phospho akt
    E2F1 abrogates Wnt signaling by modulating <t>β-catenin</t> target gene expression and inducing the GSK3-independent degradation of β-catenin ( a ) In Saos2- TR-E2F1 cells, E2F1 represses c-myc levels without affecting the levels of TCF1 or TCF4. ( b–d ) E2F1 modulates the expression of endogenous Wnt target genes (qPCR analysis after Tet-induced E2F1 expression at 24 hours). ( e ) The kinetics of E2F1-induced AXIN2 and SIAH1 expression mirrors the E2F1 activation of CCNE1/Cyclin E and CCNA1/Cyclin A , and precedes E2F1-induced apoptosis. ( f ) E2F1 represses the expression of Wnt targets in DLD1 colorectal cancer cells. Levels of mRNA were normalized to GAPDH and the effect of E2F1 is depicted as the ratio between samples after pCMV-empty or pCMV-E2F1 (1 µg each) expression. ( g, h ) is both GSK3- and caspase-independent. Saos2 cells were treated with control (DMSO), GSK3 inhibitors (20 µM SB216763 and 5 mM LiCl), or the caspase inhibitor peptide BOC-aspartyl-FMK (BAF; 100 µM) with or without Tet-induction of E2F1 (Western blot). ( i ) Co-expression of Bcl-2 (25 ng), pRb (10–25 ng), stabilized tumor-derived β-catenin mutants (10–25 ng), or the GSK3β inhibitor SB216763 (15 uM), is sufficient to partially-rescue E2F1-induced apoptosis. Cell death was depicted as percent inhibition of EGFP loss at 48 hours after transfection with pCMV-E2F1 or pCMV-empty (100 ng each) along with EGFP expression construct. All data is expressed as mean ± s.d. (n = 3).
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    Cell Signaling Technology Inc anti phospho akt
    Activation of ERK and <t>AKT</t> pathways by 20-amino-acid OPN peptide (OPNpt20) in human umbilical vein endothelial cellS (HUVECs) and the role played by α v β 3 <t>-integrin.</t> ( a , b ) HUVECs were incubated with OPNpt20 (1 μ M ) for 30, 60, 180 or 360 min ( a ) or with 0.01, 0.1 or 1 μ M of OPNpt20 for 1 h ( b ), and total and phosphorylated-ERK and -AKT levels were assessed by immunoblotting. ( c ) HUVECs were incubated with OPNpt20 (1 μ M ) or its three mutant peptides for 30 min in the presence or absence of anti-α v β 3 antibody or IgG, and total and phosphorylated-ERK and -AKT levels were assessed by immunoblotting. Representative images are presented, and results are presented as the mean±s.e.m. ( n =3). * P
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    Cell Signaling Technology Inc p akt
    POSTN promoted the stemness of heat-exposed residual HCC cells via integrin <t>β1/AKT/GSK-3β/β-catenin/TCF4/Nanog</t> signaling pathway. Western blots were performed. ( a ) POSTN upregulated the phosphorylation level of AKT and GSK-3β, and the expression of nuclear β-catenin, TCF4 and Nanog in heat-exposed residual MHCC97H and Huh7 cells. The levels of GSK-3β phosphorylation, β-catenin, TCF4 and Nanog expression were reduced by the administration of GSK-3β inhibitor CHIR 99021. ( b ) Integrin β1 knockdown attenuated POSTN-induced levels of AKT and GSK-3β phosphorylation, β-catenin, TCF4 and Nanog expression in heat-exposed residual MHCC97H cells.
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    Cell Signaling Technology Inc anti vascular cell adhesion molecule 1
    Digoxin inhibits platelet-derived growth factor (PDGF)-BB-induced adhesion molecule expression and effects the expression of key proteins in the extracelluar matrix in vascular smooth muscle cells (VSMCs). VSMCs were pre-cultured in serum-free medium for 24 h. The serum-starved VSMCs were then stimulated with PDGF-BB for 48 h in the absence or presence of digoxin (100 nM). The protein levels of intercellular adhesion <t>molecule-1</t> (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by western blot analysis. (A) One representative image out of 4 independently performed experiments is shown. (B–D) The graphs represented the relative level of these proteins for 4 independent experiment. ( # P
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    Cell Signaling Technology Inc anti β actin
    Digoxin inhibits platelet-derived growth factor (PDGF)-BB-induced adhesion molecule expression and effects the expression of key proteins in the extracelluar matrix in vascular smooth muscle cells (VSMCs). VSMCs were pre-cultured in serum-free medium for 24 h. The serum-starved VSMCs were then stimulated with PDGF-BB for 48 h in the absence or presence of digoxin (100 nM). The protein levels of intercellular adhesion <t>molecule-1</t> (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by western blot analysis. (A) One representative image out of 4 independently performed experiments is shown. (B–D) The graphs represented the relative level of these proteins for 4 independent experiment. ( # P
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    pi3k  (Abcam)
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    Abcam pi3k
    Western blot analysis for expressions of Integrin β1, FAK, <t>PI3K,</t> AKT, GSK-3β, CyclinD1, BAD in nude mice model. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P
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    Bethyl a302 021a
    Western blot analysis for expressions of Integrin β1, FAK, <t>PI3K,</t> AKT, GSK-3β, CyclinD1, BAD in nude mice model. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P
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    Cell Signaling Technology Inc anti active β catenin
    Co‐culture with Angptl2 −/− ISEMF s decreases organoid formation in vitro Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n = 4. Data from vehicle/Wnt (+) were set at 1. Schematic illustration of co‐culture of colon organoids with ISEMFs in direct contact. Colon organoid cultures in the presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm. Organoid growth efficiency and size in the presence of ISEMFs with or without Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs/Nog (+) were set at 1. Model of ANGPTL2 activity in the intestinal stem cell niche. ISEMF‐derived ANGPTL2 inhibits BMP signaling in an autocrine manner through the integrin α5β1/NF‐κB pathway to maintain ISC stemness by regulating <t>β‐catenin</t> and Smad 1/5 signaling. Data information: Data are represented as mean ± SEM. * P
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    Co‐culture with Angptl2 −/− ISEMF s decreases organoid formation in vitro Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n = 4. Data from vehicle/Wnt (+) were set at 1. Schematic illustration of co‐culture of colon organoids with ISEMFs in direct contact. Colon organoid cultures in the presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm. Organoid growth efficiency and size in the presence of ISEMFs with or without Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs/Nog (+) were set at 1. Model of ANGPTL2 activity in the intestinal stem cell niche. ISEMF‐derived ANGPTL2 inhibits BMP signaling in an autocrine manner through the integrin α5β1/NF‐κB pathway to maintain ISC stemness by regulating <t>β‐catenin</t> and Smad 1/5 signaling. Data information: Data are represented as mean ± SEM. * P
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    Millipore anti β catenin
    sFRP-1 induction of EC spreading is independent of <t>β-catenin</t> and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated
    Anti β Catenin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti alpha actinin sarcomeric antibody mouse monoclonal
    sFRP-1 induction of EC spreading is independent of <t>β-catenin</t> and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated
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    Millipore anti β1 integrin
    <t>Beta1-integrin</t> is degraded and locates to the perinuclear region. All treatments were performed with 4.3 μM LecB. (A) Cells were treated as indicated for different time periods and <t>β1-integrin</t> protein levels were determined by Western blot analysis and densitometric quantification using ImageJ. Protein levels were normalized to actin. Representative blots (above) and quantification data (below) are depicted. Values represent the mean of at least three independent experiments ± SEM. Asterisks indicate the statistical significance. (B) Cells were treated as indicated for 1 h and 5 h and analyzed by confocal fluorescence microscopy with immunostainings for β-catenin (green), β1-integrin (red) and counterstaining for DNA (DAPI, blue).
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    Millipore gfp vector
    PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were <t>transfected</t> into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The <t>GFP-RUFY3</t> and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot
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    PAK1 positively regulates <t>RUFY3</t> expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The <t>GFP-RUFY3</t> and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot
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    Millipore anti α sma
    PAK1 positively regulates <t>RUFY3</t> expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The <t>GFP-RUFY3</t> and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot
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    Cell Signaling Technology Inc gsk 3β
    PAK1 positively regulates <t>RUFY3</t> expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The <t>GFP-RUFY3</t> and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot
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    Expression levels of THBS2 in tissue samples of AAA patients and organ donors (CTL) quantified by Western Blot. ( A ) Bands obtained in Western blot analysis of THBS2 in AAA and CTL tissue samples (representative image of three samples of each group); ( B ) Bar graph of expression levels of THBS2 in tissue specimens of AAA ( n = 11) and CTL ( n = 7) group calculated as a relative expression using <t>α-tubulin</t> for normalization; ( C ) Correlation of log (THBS2/α-tubulin) levels and miRNA-195-5p (2 −∆Ct ) expression in tissue samples ( n = 18, CTL and AAA). *, p
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    Cell Signaling Technology Inc phospho mtor ser2481
    mTORC2 regulates stemness of GBM stem-like cells. ( a ) Representative immunoblot and coimmunoprecipitation demonstrated higher mTORC2 activity and formation as evidenced by increased <t>mTOR</t> <t>Ser2481</t> phosphorylation and association with Rictor in CD133-positive stem-like cells derived from U87MG. APC-conjugated CD133 antibody was from Mylteni Biotech. ( b ) Immunoblot analysis showing enhanced mTOR Ser2481 and Gli2 FL in GBM stem-like cells compared with normal human glial stem cells. ( c ) Rictor was overexpressed in LN229 cells. Simultaneously, Gli2 was knocked down in Rictor-overexpressed LN229 cells and cultured in stem cell medium. Rictor-overexpressed cells showed enhanced neurosphere formation but were unable to form large colonies when Gli2 was knocked down. ( d ) Both the number of CD133-positive cells and their expression were higher in Rictor-overexpressed LN229 cells as revealed by flow cytometric analysis. ( e ) Representative western blots showed increased levels of Oct4, Sox2, Nanog, Integrin α 6 and Nestin in Rictor-overexpressed LN229 cells. Nestin antibody was from Biolegend. Statistical significance compared with the control is indicated by * P
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    Image Search Results


    Overview of the mechanism by which MMP-9 regulates formation of focal adhesion junctions. (A) During exocytosis and upon being secreted into the extracellular space, proMMP-9 forms a large complex with itself that can then act as a scaffold for promoting outside-in signaling. In our model, PEX-9 scaffolding promotes the association of β1 integrin-EGFR-CD44. Interaction with CD44 results in enhanced EGFR activation in addition to increased phosphorylation of its downstream targets AKT and Erk 1 + 2 (through the MAPK/Erk pathway). While complexed to β1 integrin, EGFR then goes on to transactivate Src kinase after it is recruited to the α4 integrin subunit during the generation of a new focal adhesion contact site. After activation, Src can then directly phosphorylate FAK currently associated with the β1 integrin subunit resulting in its maximal catalytic activity. This active Src–FAK complex can then bind and activate PAX, resulting in a mature FAK-PAX complex necessary for the formation of focal adhesion junctions. Formation of a complex between FAK and PAX results in final translocation to ECM–integrin junctions at the cell surface where they regulate cytoskeletal interactions resulting in enhanced cellular adhesion, migration, and invasion of cancer cells. (B) Treatment with PEX-9 inhibitor (depicted as red triangle) prevents MMP-9 scaffolding, thereby preventing downstream signaling driven by EGFR activation.

    Journal: ACS Chemical Biology

    Article Title: Targeting the Hemopexin-like Domain of Latent Matrix Metalloproteinase-9 (proMMP-9) with a Small Molecule Inhibitor Prevents the Formation of Focal Adhesion Junctions

    doi: 10.1021/acschembio.7b00758

    Figure Lengend Snippet: Overview of the mechanism by which MMP-9 regulates formation of focal adhesion junctions. (A) During exocytosis and upon being secreted into the extracellular space, proMMP-9 forms a large complex with itself that can then act as a scaffold for promoting outside-in signaling. In our model, PEX-9 scaffolding promotes the association of β1 integrin-EGFR-CD44. Interaction with CD44 results in enhanced EGFR activation in addition to increased phosphorylation of its downstream targets AKT and Erk 1 + 2 (through the MAPK/Erk pathway). While complexed to β1 integrin, EGFR then goes on to transactivate Src kinase after it is recruited to the α4 integrin subunit during the generation of a new focal adhesion contact site. After activation, Src can then directly phosphorylate FAK currently associated with the β1 integrin subunit resulting in its maximal catalytic activity. This active Src–FAK complex can then bind and activate PAX, resulting in a mature FAK-PAX complex necessary for the formation of focal adhesion junctions. Formation of a complex between FAK and PAX results in final translocation to ECM–integrin junctions at the cell surface where they regulate cytoskeletal interactions resulting in enhanced cellular adhesion, migration, and invasion of cancer cells. (B) Treatment with PEX-9 inhibitor (depicted as red triangle) prevents MMP-9 scaffolding, thereby preventing downstream signaling driven by EGFR activation.

    Article Snippet: Rabbit anti-p-AKTSer473 , anti-AKT (total), anti-β1 integrin, anti-p-Erk1/2Thr202/Tyr204 , anti-p-FAKTyr 576/577 , anti-FAK (total), anti-p-PAXTyr118 , anti-PAX (total), and anti-EGFR (total) antibodies were all purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Activated Clotting Time Assay, Scaffolding, Activation Assay, Activity Assay, Translocation Assay, Migration

    E2F1 abrogates Wnt signaling by modulating β-catenin target gene expression and inducing the GSK3-independent degradation of β-catenin ( a ) In Saos2- TR-E2F1 cells, E2F1 represses c-myc levels without affecting the levels of TCF1 or TCF4. ( b–d ) E2F1 modulates the expression of endogenous Wnt target genes (qPCR analysis after Tet-induced E2F1 expression at 24 hours). ( e ) The kinetics of E2F1-induced AXIN2 and SIAH1 expression mirrors the E2F1 activation of CCNE1/Cyclin E and CCNA1/Cyclin A , and precedes E2F1-induced apoptosis. ( f ) E2F1 represses the expression of Wnt targets in DLD1 colorectal cancer cells. Levels of mRNA were normalized to GAPDH and the effect of E2F1 is depicted as the ratio between samples after pCMV-empty or pCMV-E2F1 (1 µg each) expression. ( g, h ) is both GSK3- and caspase-independent. Saos2 cells were treated with control (DMSO), GSK3 inhibitors (20 µM SB216763 and 5 mM LiCl), or the caspase inhibitor peptide BOC-aspartyl-FMK (BAF; 100 µM) with or without Tet-induction of E2F1 (Western blot). ( i ) Co-expression of Bcl-2 (25 ng), pRb (10–25 ng), stabilized tumor-derived β-catenin mutants (10–25 ng), or the GSK3β inhibitor SB216763 (15 uM), is sufficient to partially-rescue E2F1-induced apoptosis. Cell death was depicted as percent inhibition of EGFP loss at 48 hours after transfection with pCMV-E2F1 or pCMV-empty (100 ng each) along with EGFP expression construct. All data is expressed as mean ± s.d. (n = 3).

    Journal: Nature

    Article Title: E2F1 represses ?-catenin transcription and is antagonized by both pRB and CDK8

    doi: 10.1038/nature07310

    Figure Lengend Snippet: E2F1 abrogates Wnt signaling by modulating β-catenin target gene expression and inducing the GSK3-independent degradation of β-catenin ( a ) In Saos2- TR-E2F1 cells, E2F1 represses c-myc levels without affecting the levels of TCF1 or TCF4. ( b–d ) E2F1 modulates the expression of endogenous Wnt target genes (qPCR analysis after Tet-induced E2F1 expression at 24 hours). ( e ) The kinetics of E2F1-induced AXIN2 and SIAH1 expression mirrors the E2F1 activation of CCNE1/Cyclin E and CCNA1/Cyclin A , and precedes E2F1-induced apoptosis. ( f ) E2F1 represses the expression of Wnt targets in DLD1 colorectal cancer cells. Levels of mRNA were normalized to GAPDH and the effect of E2F1 is depicted as the ratio between samples after pCMV-empty or pCMV-E2F1 (1 µg each) expression. ( g, h ) is both GSK3- and caspase-independent. Saos2 cells were treated with control (DMSO), GSK3 inhibitors (20 µM SB216763 and 5 mM LiCl), or the caspase inhibitor peptide BOC-aspartyl-FMK (BAF; 100 µM) with or without Tet-induction of E2F1 (Western blot). ( i ) Co-expression of Bcl-2 (25 ng), pRb (10–25 ng), stabilized tumor-derived β-catenin mutants (10–25 ng), or the GSK3β inhibitor SB216763 (15 uM), is sufficient to partially-rescue E2F1-induced apoptosis. Cell death was depicted as percent inhibition of EGFP loss at 48 hours after transfection with pCMV-E2F1 or pCMV-empty (100 ng each) along with EGFP expression construct. All data is expressed as mean ± s.d. (n = 3).

    Article Snippet: Other antibodies used include dE2F1 (polyclonal anti-rabbit, Carol Seum), E2F1 (Santa Cruz, sc-193), E2F4 (Santa Cruz, sc-1082), DP1 (Santa Cruz, sc-610), CRSP70 (Santa Cruz, sc-9426), c-myc (Santa Cruz, sc-40), TCF1 (Santa Cruz, sc-8589), TCF4 (Santa Cruz, sc-8632; Upstate, 05–511 for ChIP), Cyclin E (Santa Cruz, sc-247), Cyclin A (Santa Cruz, sc-596), pan-MAPK (BD Biosciences, 612641), β-catenin (Cell Signaling, 9562), CDK8 (Santa Cruz, sc-1521), pRB (Santa Cruz, sc-50), Arm (N27A1; Developmental Studies Hybridoma Bank), β1-integrin (BD Biosciences, 610467), E-cadherin (BD Biosciences, 610181), N-cadherin (BD Biosciences, 610920), α-catenin (BD Biosciences 610193), FAK (BD Biosciences 610087), phospho-FAK Tyr397 (BD Biosciences, 611806), GSK3 (Cell Signaling, 9315), phospho-GSK3 (Upstate, 05–413), Src (Cell Signaling, 2108), phospho-Src Tyr416 (Cell Signaling, 2101), STAT3 (Cell Signaling, 9132), phospho-STAT3 Tyr705 (Cell Signaling, 9131), phospho-STAT3 Ser727 (Cell Signaling, 9134), STAT5 (Cell Signaling, 9310), phospho-STAT5 Tyr694 (Cell Signaling, 9356), SHC (BD Biosciences, 610081), anti-HA-epitope (Sigma, H6908), and GFP (Sigma, G1544).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Western Blot, Derivative Assay, Inhibition, Transfection, Construct

    Functional antagonism between E2F1 and β-catenin/TCF-signaling ( a ) Normal Drosophila wing of Act88F-Gal4/+ genotype. A dE2F1 -induced apoptotic wing phenotype ( b ) is strongly suppressed by co-expression of arm ( d ) or pan/dTCF ( e ), phenocopied by expression of dominant-negative dTCF (dTCFΔN) ( f ), and strongly synergizes with sgg/GSK3 co-expression ( h ). Expression of arm ( c ), sgg ( g ), or pan (not shown) alone does not induce a wing phenotype. ( i–l ) GMR -mediated eye-specific expression of dE2F1/dDP strongly suppresses a rough eye phenotype induced by activated- arm (arm*; S44Y) expression. All phenotypes were compared in female progeny from F1 crosses conducted at 25°C versus control ( w 1118 background). ( m ) E2F1 expression activates a canonical pE2F4B-luciferase reporter while abrogating the S33Y-β-catenin-mediated activation of pTopFLASH. ( n ) Inhibition of pTopFLASH activity is specific to E2F1. ( o ) E2F1 dominantly inhibits pTopFLASH activation by p73. All data is expressed as mean ± s.d. (n = 3) of normalized relative light units (NRLU) of luciferase.

    Journal: Nature

    Article Title: E2F1 represses ?-catenin transcription and is antagonized by both pRB and CDK8

    doi: 10.1038/nature07310

    Figure Lengend Snippet: Functional antagonism between E2F1 and β-catenin/TCF-signaling ( a ) Normal Drosophila wing of Act88F-Gal4/+ genotype. A dE2F1 -induced apoptotic wing phenotype ( b ) is strongly suppressed by co-expression of arm ( d ) or pan/dTCF ( e ), phenocopied by expression of dominant-negative dTCF (dTCFΔN) ( f ), and strongly synergizes with sgg/GSK3 co-expression ( h ). Expression of arm ( c ), sgg ( g ), or pan (not shown) alone does not induce a wing phenotype. ( i–l ) GMR -mediated eye-specific expression of dE2F1/dDP strongly suppresses a rough eye phenotype induced by activated- arm (arm*; S44Y) expression. All phenotypes were compared in female progeny from F1 crosses conducted at 25°C versus control ( w 1118 background). ( m ) E2F1 expression activates a canonical pE2F4B-luciferase reporter while abrogating the S33Y-β-catenin-mediated activation of pTopFLASH. ( n ) Inhibition of pTopFLASH activity is specific to E2F1. ( o ) E2F1 dominantly inhibits pTopFLASH activation by p73. All data is expressed as mean ± s.d. (n = 3) of normalized relative light units (NRLU) of luciferase.

    Article Snippet: Other antibodies used include dE2F1 (polyclonal anti-rabbit, Carol Seum), E2F1 (Santa Cruz, sc-193), E2F4 (Santa Cruz, sc-1082), DP1 (Santa Cruz, sc-610), CRSP70 (Santa Cruz, sc-9426), c-myc (Santa Cruz, sc-40), TCF1 (Santa Cruz, sc-8589), TCF4 (Santa Cruz, sc-8632; Upstate, 05–511 for ChIP), Cyclin E (Santa Cruz, sc-247), Cyclin A (Santa Cruz, sc-596), pan-MAPK (BD Biosciences, 612641), β-catenin (Cell Signaling, 9562), CDK8 (Santa Cruz, sc-1521), pRB (Santa Cruz, sc-50), Arm (N27A1; Developmental Studies Hybridoma Bank), β1-integrin (BD Biosciences, 610467), E-cadherin (BD Biosciences, 610181), N-cadherin (BD Biosciences, 610920), α-catenin (BD Biosciences 610193), FAK (BD Biosciences 610087), phospho-FAK Tyr397 (BD Biosciences, 611806), GSK3 (Cell Signaling, 9315), phospho-GSK3 (Upstate, 05–413), Src (Cell Signaling, 2108), phospho-Src Tyr416 (Cell Signaling, 2101), STAT3 (Cell Signaling, 9132), phospho-STAT3 Tyr705 (Cell Signaling, 9131), phospho-STAT3 Ser727 (Cell Signaling, 9134), STAT5 (Cell Signaling, 9310), phospho-STAT5 Tyr694 (Cell Signaling, 9356), SHC (BD Biosciences, 610081), anti-HA-epitope (Sigma, H6908), and GFP (Sigma, G1544).

    Techniques: Functional Assay, Expressing, Dominant Negative Mutation, Luciferase, Activation Assay, Inhibition, Activity Assay

    pRB inactivation abrogates β-catenin/TCF-dependent transcription ( a–b )High levels of pRB and β-catenin colocalize within the tumor epithelium of an Apc Min colonic tumor. ( c ) Endogenous pRB was depleted for 6 days in U2OS- shRb cells (containing DOX-inducible short-hairpin- Rb and GFP transgenes) ( d ) pRB depletion activates E2F and represses TCF activity. Basal and activated E2F and TCF activity was determined by transfection of their respective luciferase reporter plasmids (for 24 hours) in the absence (basal) or presence (activated) of E2F1 or S33Y-β-catenin expression constructs. ( e ) In SW480 colorectal cancer cells, E2F1 expression is sufficient to activate E2F activity and repress endogenous β-catenin activity in a dose-dependent manner. ( f ) Expression of short-hairpin pRB (400 ng) is sufficient to repress pTopFLASH activity in three different colorectal cancer cell lines (SW480, DLD1, and HCT116). ( g ) pRB inactivation reduces clonogenic survival of SW480 cells and is partially rescued by S33Y-β-catenin/TCF1E , but not Bcl-2 expression. SW480 cells were Amaxa nucleoporated with either control LLP-GFP or - Rb silencing constructs (1 µg each) with or without S33Y-β-catenin (250 ng), TCF1E (250 ng), or Bcl-2 (500 ng) expression constructs, and survival was determined at day 5 by MTT assay. All data is expressed as mean ± s.d. (n = 3; *p

    Journal: Nature

    Article Title: E2F1 represses ?-catenin transcription and is antagonized by both pRB and CDK8

    doi: 10.1038/nature07310

    Figure Lengend Snippet: pRB inactivation abrogates β-catenin/TCF-dependent transcription ( a–b )High levels of pRB and β-catenin colocalize within the tumor epithelium of an Apc Min colonic tumor. ( c ) Endogenous pRB was depleted for 6 days in U2OS- shRb cells (containing DOX-inducible short-hairpin- Rb and GFP transgenes) ( d ) pRB depletion activates E2F and represses TCF activity. Basal and activated E2F and TCF activity was determined by transfection of their respective luciferase reporter plasmids (for 24 hours) in the absence (basal) or presence (activated) of E2F1 or S33Y-β-catenin expression constructs. ( e ) In SW480 colorectal cancer cells, E2F1 expression is sufficient to activate E2F activity and repress endogenous β-catenin activity in a dose-dependent manner. ( f ) Expression of short-hairpin pRB (400 ng) is sufficient to repress pTopFLASH activity in three different colorectal cancer cell lines (SW480, DLD1, and HCT116). ( g ) pRB inactivation reduces clonogenic survival of SW480 cells and is partially rescued by S33Y-β-catenin/TCF1E , but not Bcl-2 expression. SW480 cells were Amaxa nucleoporated with either control LLP-GFP or - Rb silencing constructs (1 µg each) with or without S33Y-β-catenin (250 ng), TCF1E (250 ng), or Bcl-2 (500 ng) expression constructs, and survival was determined at day 5 by MTT assay. All data is expressed as mean ± s.d. (n = 3; *p

    Article Snippet: Other antibodies used include dE2F1 (polyclonal anti-rabbit, Carol Seum), E2F1 (Santa Cruz, sc-193), E2F4 (Santa Cruz, sc-1082), DP1 (Santa Cruz, sc-610), CRSP70 (Santa Cruz, sc-9426), c-myc (Santa Cruz, sc-40), TCF1 (Santa Cruz, sc-8589), TCF4 (Santa Cruz, sc-8632; Upstate, 05–511 for ChIP), Cyclin E (Santa Cruz, sc-247), Cyclin A (Santa Cruz, sc-596), pan-MAPK (BD Biosciences, 612641), β-catenin (Cell Signaling, 9562), CDK8 (Santa Cruz, sc-1521), pRB (Santa Cruz, sc-50), Arm (N27A1; Developmental Studies Hybridoma Bank), β1-integrin (BD Biosciences, 610467), E-cadherin (BD Biosciences, 610181), N-cadherin (BD Biosciences, 610920), α-catenin (BD Biosciences 610193), FAK (BD Biosciences 610087), phospho-FAK Tyr397 (BD Biosciences, 611806), GSK3 (Cell Signaling, 9315), phospho-GSK3 (Upstate, 05–413), Src (Cell Signaling, 2108), phospho-Src Tyr416 (Cell Signaling, 2101), STAT3 (Cell Signaling, 9132), phospho-STAT3 Tyr705 (Cell Signaling, 9131), phospho-STAT3 Ser727 (Cell Signaling, 9134), STAT5 (Cell Signaling, 9310), phospho-STAT5 Tyr694 (Cell Signaling, 9356), SHC (BD Biosciences, 610081), anti-HA-epitope (Sigma, H6908), and GFP (Sigma, G1544).

    Techniques: Activity Assay, Transfection, Luciferase, Expressing, Construct, MTT Assay

    CDK8 antagonizes E2F1 activity ( a–c )The dCdk8 c01804 mutant dominantly suppresses a rough eye phenotype caused by the eye-specific expression of a dE2F1 RNAi transgene. ( d ) The expression of the dE2F1 target genes, PCNA and MCM5 are upregulated in dCdk8 (null or hypomorphic alleles) or dCycC null mutant Drosophila larvae, while the expression of the dE2F2 target gene vasa is unaffected. ( e ) dCDK8 physically interacts with dE2F1 by GST-pulldown assay. ( f ) Co-immunoprecipitation of human E2F1 and CDK8 from Saos2 whole-cell extracts. As control, E2F1 does not associate with the CRSP70 small-Mediator subunit. ( g ) CDK8 binds to and specifically phosphorylates E2F1. Kinase-assay was performed following CDK8 or CRSP70 (as control) immunoprecipitation from Saos2 cells. E2F1, E2F4, or DP1 were re-immunoprecipitated and resolved in 12% SDS-PAGE. ( h ) The expression of wild-type CDK8, but not kinase-dead, abrogates the inhibitory effects of E2F1 on β-catenin/TCF-dependent transcription. pTopFLASH assays were determined at 48 hours in co-transfection experiments with 200 ng CDK8 or CDK8KD in Saos2 cells. ( i ) Expression of short-hairpin pRB (400 ng) and CDK8KD (300 ng) co-operatively repress pTopFLASH activity in SW480 colorectal cancer cells. All data is expressed as mean ± s.d. (n = 3).

    Journal: Nature

    Article Title: E2F1 represses ?-catenin transcription and is antagonized by both pRB and CDK8

    doi: 10.1038/nature07310

    Figure Lengend Snippet: CDK8 antagonizes E2F1 activity ( a–c )The dCdk8 c01804 mutant dominantly suppresses a rough eye phenotype caused by the eye-specific expression of a dE2F1 RNAi transgene. ( d ) The expression of the dE2F1 target genes, PCNA and MCM5 are upregulated in dCdk8 (null or hypomorphic alleles) or dCycC null mutant Drosophila larvae, while the expression of the dE2F2 target gene vasa is unaffected. ( e ) dCDK8 physically interacts with dE2F1 by GST-pulldown assay. ( f ) Co-immunoprecipitation of human E2F1 and CDK8 from Saos2 whole-cell extracts. As control, E2F1 does not associate with the CRSP70 small-Mediator subunit. ( g ) CDK8 binds to and specifically phosphorylates E2F1. Kinase-assay was performed following CDK8 or CRSP70 (as control) immunoprecipitation from Saos2 cells. E2F1, E2F4, or DP1 were re-immunoprecipitated and resolved in 12% SDS-PAGE. ( h ) The expression of wild-type CDK8, but not kinase-dead, abrogates the inhibitory effects of E2F1 on β-catenin/TCF-dependent transcription. pTopFLASH assays were determined at 48 hours in co-transfection experiments with 200 ng CDK8 or CDK8KD in Saos2 cells. ( i ) Expression of short-hairpin pRB (400 ng) and CDK8KD (300 ng) co-operatively repress pTopFLASH activity in SW480 colorectal cancer cells. All data is expressed as mean ± s.d. (n = 3).

    Article Snippet: Other antibodies used include dE2F1 (polyclonal anti-rabbit, Carol Seum), E2F1 (Santa Cruz, sc-193), E2F4 (Santa Cruz, sc-1082), DP1 (Santa Cruz, sc-610), CRSP70 (Santa Cruz, sc-9426), c-myc (Santa Cruz, sc-40), TCF1 (Santa Cruz, sc-8589), TCF4 (Santa Cruz, sc-8632; Upstate, 05–511 for ChIP), Cyclin E (Santa Cruz, sc-247), Cyclin A (Santa Cruz, sc-596), pan-MAPK (BD Biosciences, 612641), β-catenin (Cell Signaling, 9562), CDK8 (Santa Cruz, sc-1521), pRB (Santa Cruz, sc-50), Arm (N27A1; Developmental Studies Hybridoma Bank), β1-integrin (BD Biosciences, 610467), E-cadherin (BD Biosciences, 610181), N-cadherin (BD Biosciences, 610920), α-catenin (BD Biosciences 610193), FAK (BD Biosciences 610087), phospho-FAK Tyr397 (BD Biosciences, 611806), GSK3 (Cell Signaling, 9315), phospho-GSK3 (Upstate, 05–413), Src (Cell Signaling, 2108), phospho-Src Tyr416 (Cell Signaling, 2101), STAT3 (Cell Signaling, 9132), phospho-STAT3 Tyr705 (Cell Signaling, 9131), phospho-STAT3 Ser727 (Cell Signaling, 9134), STAT5 (Cell Signaling, 9310), phospho-STAT5 Tyr694 (Cell Signaling, 9356), SHC (BD Biosciences, 610081), anti-HA-epitope (Sigma, H6908), and GFP (Sigma, G1544).

    Techniques: Activity Assay, Mutagenesis, Expressing, GST Pulldown Assay, Immunoprecipitation, Kinase Assay, SDS Page, Cotransfection

    Activation of ERK and AKT pathways by 20-amino-acid OPN peptide (OPNpt20) in human umbilical vein endothelial cellS (HUVECs) and the role played by α v β 3 -integrin. ( a , b ) HUVECs were incubated with OPNpt20 (1 μ M ) for 30, 60, 180 or 360 min ( a ) or with 0.01, 0.1 or 1 μ M of OPNpt20 for 1 h ( b ), and total and phosphorylated-ERK and -AKT levels were assessed by immunoblotting. ( c ) HUVECs were incubated with OPNpt20 (1 μ M ) or its three mutant peptides for 30 min in the presence or absence of anti-α v β 3 antibody or IgG, and total and phosphorylated-ERK and -AKT levels were assessed by immunoblotting. Representative images are presented, and results are presented as the mean±s.e.m. ( n =3). * P

    Journal: Experimental & Molecular Medicine

    Article Title: Proangiogenic functions of an RGD-SLAY-containing osteopontin icosamer peptide in HUVECs and in the postischemic brain

    doi: 10.1038/emm.2017.241

    Figure Lengend Snippet: Activation of ERK and AKT pathways by 20-amino-acid OPN peptide (OPNpt20) in human umbilical vein endothelial cellS (HUVECs) and the role played by α v β 3 -integrin. ( a , b ) HUVECs were incubated with OPNpt20 (1 μ M ) for 30, 60, 180 or 360 min ( a ) or with 0.01, 0.1 or 1 μ M of OPNpt20 for 1 h ( b ), and total and phosphorylated-ERK and -AKT levels were assessed by immunoblotting. ( c ) HUVECs were incubated with OPNpt20 (1 μ M ) or its three mutant peptides for 30 min in the presence or absence of anti-α v β 3 antibody or IgG, and total and phosphorylated-ERK and -AKT levels were assessed by immunoblotting. Representative images are presented, and results are presented as the mean±s.e.m. ( n =3). * P

    Article Snippet: Cell or tissue extracts were then loaded into 8–10% SDS-PAGE gels and immunoblotted using the following primary antibodies: anti-vascular endothelial growth factor (VEGF) (1:3000; Abcam, Cambridge, UK), anti-α-smooth muscle actin (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-matrix metallopeptidase 9 (MMP9) (1:3000, GeneTex, Irvine, CA, USA), anti-endothelial NOS (eNOS) (1:3000; Santa Cruz Biotechnology), anti-phospho-eNOS (1:3000; Cell Signaling Technology, Danvers, MA, USA), anti-neuronal NOS (nNOS) (1:3000; Santa Cruz Biotechnology), anti-phospho-nNOS (1:3000; Cell Signaling Technology), anti-integrin α4 , anti-integrin α9 , anti-integrin αv , anti-integrin-β3 (1:2000; Santa Cruz Biotechnology), anti-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), anti-phospho-PI3K, anti-ERK, anti-phospho-ERK, anti-Akt, anti-phospho-Akt (1:3000; Cell Signaling) or anti-β-actin (1:4000; Applied Biological Materials, Richmond, BC, Canada) antibody.

    Techniques: Activation Assay, Incubation, Mutagenesis

    POSTN promoted the stemness of heat-exposed residual HCC cells via integrin β1/AKT/GSK-3β/β-catenin/TCF4/Nanog signaling pathway. Western blots were performed. ( a ) POSTN upregulated the phosphorylation level of AKT and GSK-3β, and the expression of nuclear β-catenin, TCF4 and Nanog in heat-exposed residual MHCC97H and Huh7 cells. The levels of GSK-3β phosphorylation, β-catenin, TCF4 and Nanog expression were reduced by the administration of GSK-3β inhibitor CHIR 99021. ( b ) Integrin β1 knockdown attenuated POSTN-induced levels of AKT and GSK-3β phosphorylation, β-catenin, TCF4 and Nanog expression in heat-exposed residual MHCC97H cells.

    Journal: Scientific Reports

    Article Title: Activated hepatic stellate cells secrete periostin to induce stem cell-like phenotype of residual hepatocellular carcinoma cells after heat treatment

    doi: 10.1038/s41598-017-01177-6

    Figure Lengend Snippet: POSTN promoted the stemness of heat-exposed residual HCC cells via integrin β1/AKT/GSK-3β/β-catenin/TCF4/Nanog signaling pathway. Western blots were performed. ( a ) POSTN upregulated the phosphorylation level of AKT and GSK-3β, and the expression of nuclear β-catenin, TCF4 and Nanog in heat-exposed residual MHCC97H and Huh7 cells. The levels of GSK-3β phosphorylation, β-catenin, TCF4 and Nanog expression were reduced by the administration of GSK-3β inhibitor CHIR 99021. ( b ) Integrin β1 knockdown attenuated POSTN-induced levels of AKT and GSK-3β phosphorylation, β-catenin, TCF4 and Nanog expression in heat-exposed residual MHCC97H cells.

    Article Snippet: The equal amounts of separated proteins (20 μg) were transferred onto PVDF membranes (Millipore, USA) and incubated with primary antibodies against POSTN (1:1000, Abcam), collagen I (1:1000, Abcam), α-SMA (1:300, Abcam), AMPK α (1:1000, CST, Cell Signal Technology), p-AMPK α (1:1000, CST), AKT (1:1000, CST), p-AKT (Ser 473) (1:2000, CST), GSK-3β (1:1000, CST), p-GSK-3β (1:1000, Tyr 216) (Abcam), integrin β1(1:1000, CST), TCF4 (1:1000, CST), β-catenin (1:1000, CST), Nanog (1:2000, CST), β-actin (1:1000, Beyotime), Tubulin (1:1000, Beyotime), Lamin B1 (1:1000, InTech) and corresponding HRP-conjugated secondary antibodies (Jackson).

    Techniques: Western Blot, Expressing

    Metformin suppressed tumor progression of heat-exposed residual HCC cells by inhibiting POSTN secretion from activated HSCs and down-regulation of cancer stem cell markers. ( a , b , c ) Metformin inhibited α-SMA, collagen type 1 alpha 1 (COL1A1) and POSTN mRNA expression in the activated HSCs and decreased the supernatant concentration of POSTN in the HSC-CM. Quantitative RT-PCR and ELISA were performed. POSTN were down-regulated by metformin in dose- and time-dependent manner through p-AMPK activation and p-AKT inhibition, which was reversed by AMPK inhibitor Compound C. Western blots were performed. Nanog expression was decreased in residual MHCC97H and Huh7 cells co-cultured with CM from metformin-treated pHSCs (primary human hepatic stellate cells) compared to with control medium. ( d ) Heat-exposed residual MHCC97H cell co-inoculated with pHSCs subcutaneously in the right flank of nude mice (n = 6 mice per group). The in vivo tumor growth generated from heat-exposed residual HCC cells with activated HSCs was significantly inhibited in the metformin-treated group. The lower expression of Nanog, CD133, EpCAM and POSTN in the metformin-treated tumors was detected by quantitative RT-PCR and immunohistochemical analysis. ** P

    Journal: Scientific Reports

    Article Title: Activated hepatic stellate cells secrete periostin to induce stem cell-like phenotype of residual hepatocellular carcinoma cells after heat treatment

    doi: 10.1038/s41598-017-01177-6

    Figure Lengend Snippet: Metformin suppressed tumor progression of heat-exposed residual HCC cells by inhibiting POSTN secretion from activated HSCs and down-regulation of cancer stem cell markers. ( a , b , c ) Metformin inhibited α-SMA, collagen type 1 alpha 1 (COL1A1) and POSTN mRNA expression in the activated HSCs and decreased the supernatant concentration of POSTN in the HSC-CM. Quantitative RT-PCR and ELISA were performed. POSTN were down-regulated by metformin in dose- and time-dependent manner through p-AMPK activation and p-AKT inhibition, which was reversed by AMPK inhibitor Compound C. Western blots were performed. Nanog expression was decreased in residual MHCC97H and Huh7 cells co-cultured with CM from metformin-treated pHSCs (primary human hepatic stellate cells) compared to with control medium. ( d ) Heat-exposed residual MHCC97H cell co-inoculated with pHSCs subcutaneously in the right flank of nude mice (n = 6 mice per group). The in vivo tumor growth generated from heat-exposed residual HCC cells with activated HSCs was significantly inhibited in the metformin-treated group. The lower expression of Nanog, CD133, EpCAM and POSTN in the metformin-treated tumors was detected by quantitative RT-PCR and immunohistochemical analysis. ** P

    Article Snippet: The equal amounts of separated proteins (20 μg) were transferred onto PVDF membranes (Millipore, USA) and incubated with primary antibodies against POSTN (1:1000, Abcam), collagen I (1:1000, Abcam), α-SMA (1:300, Abcam), AMPK α (1:1000, CST, Cell Signal Technology), p-AMPK α (1:1000, CST), AKT (1:1000, CST), p-AKT (Ser 473) (1:2000, CST), GSK-3β (1:1000, CST), p-GSK-3β (1:1000, Tyr 216) (Abcam), integrin β1(1:1000, CST), TCF4 (1:1000, CST), β-catenin (1:1000, CST), Nanog (1:2000, CST), β-actin (1:1000, Beyotime), Tubulin (1:1000, Beyotime), Lamin B1 (1:1000, InTech) and corresponding HRP-conjugated secondary antibodies (Jackson).

    Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activation Assay, Inhibition, Western Blot, Cell Culture, Mouse Assay, In Vivo, Generated, Immunohistochemistry

    Graphical diagram of the proposed mechanism by which activated HSCs induced the stemness traits in heat-exposed residual HCC cells. POSTN from activated HSCs initiates integrin β1/GSK-3β/β-catenin/TCF4 pathway to modulate stemness traits of heat-exposed residual HCC, which was reversed by metformin via the AMPK/AKT pathway to decrease POSTN secretion.

    Journal: Scientific Reports

    Article Title: Activated hepatic stellate cells secrete periostin to induce stem cell-like phenotype of residual hepatocellular carcinoma cells after heat treatment

    doi: 10.1038/s41598-017-01177-6

    Figure Lengend Snippet: Graphical diagram of the proposed mechanism by which activated HSCs induced the stemness traits in heat-exposed residual HCC cells. POSTN from activated HSCs initiates integrin β1/GSK-3β/β-catenin/TCF4 pathway to modulate stemness traits of heat-exposed residual HCC, which was reversed by metformin via the AMPK/AKT pathway to decrease POSTN secretion.

    Article Snippet: The equal amounts of separated proteins (20 μg) were transferred onto PVDF membranes (Millipore, USA) and incubated with primary antibodies against POSTN (1:1000, Abcam), collagen I (1:1000, Abcam), α-SMA (1:300, Abcam), AMPK α (1:1000, CST, Cell Signal Technology), p-AMPK α (1:1000, CST), AKT (1:1000, CST), p-AKT (Ser 473) (1:2000, CST), GSK-3β (1:1000, CST), p-GSK-3β (1:1000, Tyr 216) (Abcam), integrin β1(1:1000, CST), TCF4 (1:1000, CST), β-catenin (1:1000, CST), Nanog (1:2000, CST), β-actin (1:1000, Beyotime), Tubulin (1:1000, Beyotime), Lamin B1 (1:1000, InTech) and corresponding HRP-conjugated secondary antibodies (Jackson).

    Techniques:

    Digoxin inhibits platelet-derived growth factor (PDGF)-BB-induced adhesion molecule expression and effects the expression of key proteins in the extracelluar matrix in vascular smooth muscle cells (VSMCs). VSMCs were pre-cultured in serum-free medium for 24 h. The serum-starved VSMCs were then stimulated with PDGF-BB for 48 h in the absence or presence of digoxin (100 nM). The protein levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by western blot analysis. (A) One representative image out of 4 independently performed experiments is shown. (B–D) The graphs represented the relative level of these proteins for 4 independent experiment. ( # P

    Journal: International Journal of Molecular Medicine

    Article Title: Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury

    doi: 10.3892/ijmm.2015.2320

    Figure Lengend Snippet: Digoxin inhibits platelet-derived growth factor (PDGF)-BB-induced adhesion molecule expression and effects the expression of key proteins in the extracelluar matrix in vascular smooth muscle cells (VSMCs). VSMCs were pre-cultured in serum-free medium for 24 h. The serum-starved VSMCs were then stimulated with PDGF-BB for 48 h in the absence or presence of digoxin (100 nM). The protein levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by western blot analysis. (A) One representative image out of 4 independently performed experiments is shown. (B–D) The graphs represented the relative level of these proteins for 4 independent experiment. ( # P

    Article Snippet: The membranes were blocked, and then incubated with various antibodies overnight, such as anti-CDK4 (Cat. no. SC23896, 1:500), anti-CDK6 (Cat. no. SC53638, 1:500), anti-p27Kip1 (Cat. no. SC1641, 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SM22a (Cat. no. A5228, 1:1,000), anti-calponin (Cat. no. C2687, 1:1,000), anti-smooth muscle (SM) α-actin (Cat. no. A2172, 1:1,000) (all from Sigma), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; Tyr183/185; Cat. no. 5135, 1:1,000), anti-JNK (Cat. no. 9258 1:1,000), anti-p38 mitogen-activated protein kinase (MAPK; Cat. no. 9228, 1:1,000), anti-phospho-p38 MAPK (Thr180/Tyr182; Cat. no. 4092, 1:1,000; p-MAPK), anti-extracellular signal-regulated kinase (ERK)1/2 (Cat. no. 4696, 1:1,000), anti-phospho-ERK1/2 (p-ERK, Tyr204; Cat. no. 4374, 1:1000), anti-phospho-Akt (p-Akt, S473; Cat. no. 12694, 1:1,000), anti-Akt (Cat. no. 2920, 1:1,000), anti-phospho-GSK-3β (p-GSK-3β, S9; Cat. no. 14630, 1:1,000), anti-GSK-3β (Cat. no. 9832, 1:1,000) (all from Cell Signaling Technology), anti-ILK (Cat. no. 61183, 1:1,000; BD Biosciences, Franklin Lakes, NJ, USA), anti-intercellular adhesion molecule-1 (ICAM-1; Cat. no. 4915, 1:500), anti-vascular cell adhesion molecule-1 (VCAM-1; Cat. no. 13662, 1:500) (both from Cell Signaling Technology), anti-matrix metalloproteinase (MMP)-2 (Cat. no. SC13594, 1:500), anti-MMP-9 (Cat. no. SC21733, 1:500) anti-tissue inhibitors of metalloproteinase (TIMP)-1 (Cat. no. SC21734, 1:500), anti-TIMP-2 (Cat. no. SC365671, 1:500) (all from Santa Cruz Biotechnology, Inc.), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat. no. 5174, 1:2,500) and anti-β-actin (Cat. no. 8457, 1:2,500) (both from Cell Signaling Technology), and then with the horseradish peroxidase-conjugated secondary antibody (Beijing TDY Biotech Co., Ltd.) (1:5,000) for 2 h. Specific protein expression levels were normalized to GAPDH or β-actin for total protein analyses or to total proteins for phosphorylated protein measurements.

    Techniques: Derivative Assay, Expressing, Cell Culture, Western Blot

    Western blot analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, CyclinD1, BAD in nude mice model. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P

    Journal: Journal of Cancer

    Article Title: Chinese Herbal Medicine Wenxia Changfu Formula Reverses Cell Adhesion-Mediated Drug Resistance via the Integrin β1-PI3K-AKT Pathway in Lung Cancer

    doi: 10.7150/jca.25163

    Figure Lengend Snippet: Western blot analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, CyclinD1, BAD in nude mice model. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P

    Article Snippet: Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels electrophoresed at 200V, electrotransferred to polyvinylidene fluoride membranes, and probed with antibodies to Integrin β1(1:1000; lot number: Ab179471, Abcam, USA), FAK (1:1000; lot number: Ab40794, Abcam, USA), PI3K (1:1000; lot number: Ab40755, Abcam, USA), AKT (1:1000; lot number: Ab25893, Abcam, USA), GSK-3β (1:1000; lot number: Ab93926, Abcam, USA ), Cyclin D1 (1:1000; lot number: Ab134175, Abcam, USA), BAD (1:1000; lot number: Ab32445, Abcam, USA).

    Techniques: Western Blot, Mouse Assay

    qRT-PCR analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, Cyclin D1, BAD in nude mice model. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P

    Journal: Journal of Cancer

    Article Title: Chinese Herbal Medicine Wenxia Changfu Formula Reverses Cell Adhesion-Mediated Drug Resistance via the Integrin β1-PI3K-AKT Pathway in Lung Cancer

    doi: 10.7150/jca.25163

    Figure Lengend Snippet: qRT-PCR analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, Cyclin D1, BAD in nude mice model. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P

    Article Snippet: Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels electrophoresed at 200V, electrotransferred to polyvinylidene fluoride membranes, and probed with antibodies to Integrin β1(1:1000; lot number: Ab179471, Abcam, USA), FAK (1:1000; lot number: Ab40794, Abcam, USA), PI3K (1:1000; lot number: Ab40755, Abcam, USA), AKT (1:1000; lot number: Ab25893, Abcam, USA), GSK-3β (1:1000; lot number: Ab93926, Abcam, USA ), Cyclin D1 (1:1000; lot number: Ab134175, Abcam, USA), BAD (1:1000; lot number: Ab32445, Abcam, USA).

    Techniques: Quantitative RT-PCR, Mouse Assay

    qRT-PCR analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, Cyclin D1, BAD in control group, DDP group, combination of WCF medicated serum and DDP group and LY294002 group. Values are expressed as Mean±SD of eight mice. a P

    Journal: Journal of Cancer

    Article Title: Chinese Herbal Medicine Wenxia Changfu Formula Reverses Cell Adhesion-Mediated Drug Resistance via the Integrin β1-PI3K-AKT Pathway in Lung Cancer

    doi: 10.7150/jca.25163

    Figure Lengend Snippet: qRT-PCR analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, Cyclin D1, BAD in control group, DDP group, combination of WCF medicated serum and DDP group and LY294002 group. Values are expressed as Mean±SD of eight mice. a P

    Article Snippet: Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels electrophoresed at 200V, electrotransferred to polyvinylidene fluoride membranes, and probed with antibodies to Integrin β1(1:1000; lot number: Ab179471, Abcam, USA), FAK (1:1000; lot number: Ab40794, Abcam, USA), PI3K (1:1000; lot number: Ab40755, Abcam, USA), AKT (1:1000; lot number: Ab25893, Abcam, USA), GSK-3β (1:1000; lot number: Ab93926, Abcam, USA ), Cyclin D1 (1:1000; lot number: Ab134175, Abcam, USA), BAD (1:1000; lot number: Ab32445, Abcam, USA).

    Techniques: Quantitative RT-PCR, Mouse Assay

    Western blot analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, Cyclin D1, BAD in control group, DDP group, combination of WCF medicated serum and DDP group and LY294002 group. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P

    Journal: Journal of Cancer

    Article Title: Chinese Herbal Medicine Wenxia Changfu Formula Reverses Cell Adhesion-Mediated Drug Resistance via the Integrin β1-PI3K-AKT Pathway in Lung Cancer

    doi: 10.7150/jca.25163

    Figure Lengend Snippet: Western blot analysis for expressions of Integrin β1, FAK, PI3K, AKT, GSK-3β, Cyclin D1, BAD in control group, DDP group, combination of WCF medicated serum and DDP group and LY294002 group. The graph represents relative densitometric intensity of each band normalized to β-actin. Values are expressed as Mean±SD of eight mice. a P

    Article Snippet: Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels electrophoresed at 200V, electrotransferred to polyvinylidene fluoride membranes, and probed with antibodies to Integrin β1(1:1000; lot number: Ab179471, Abcam, USA), FAK (1:1000; lot number: Ab40794, Abcam, USA), PI3K (1:1000; lot number: Ab40755, Abcam, USA), AKT (1:1000; lot number: Ab25893, Abcam, USA), GSK-3β (1:1000; lot number: Ab93926, Abcam, USA ), Cyclin D1 (1:1000; lot number: Ab134175, Abcam, USA), BAD (1:1000; lot number: Ab32445, Abcam, USA).

    Techniques: Western Blot, Mouse Assay

    Co‐culture with Angptl2 −/− ISEMF s decreases organoid formation in vitro Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n = 4. Data from vehicle/Wnt (+) were set at 1. Schematic illustration of co‐culture of colon organoids with ISEMFs in direct contact. Colon organoid cultures in the presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm. Organoid growth efficiency and size in the presence of ISEMFs with or without Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs/Nog (+) were set at 1. Model of ANGPTL2 activity in the intestinal stem cell niche. ISEMF‐derived ANGPTL2 inhibits BMP signaling in an autocrine manner through the integrin α5β1/NF‐κB pathway to maintain ISC stemness by regulating β‐catenin and Smad 1/5 signaling. Data information: Data are represented as mean ± SEM. * P

    Journal: The EMBO Journal

    Article Title: ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis

    doi: 10.15252/embj.201695690

    Figure Lengend Snippet: Co‐culture with Angptl2 −/− ISEMF s decreases organoid formation in vitro Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n = 4. Data from vehicle/Wnt (+) were set at 1. Schematic illustration of co‐culture of colon organoids with ISEMFs in direct contact. Colon organoid cultures in the presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm. Organoid growth efficiency and size in the presence of ISEMFs with or without Noggin (Nog). n = 4. Data from wild‐type organoids/wild‐type ISEMFs/Nog (+) were set at 1. Model of ANGPTL2 activity in the intestinal stem cell niche. ISEMF‐derived ANGPTL2 inhibits BMP signaling in an autocrine manner through the integrin α5β1/NF‐κB pathway to maintain ISC stemness by regulating β‐catenin and Smad 1/5 signaling. Data information: Data are represented as mean ± SEM. * P

    Article Snippet: Nuclei were stained with DAPI for 20 min. Antibodies used were as follows: anti‐Ki67 (1:800, Leica Microsystems, #NCL‐Ki67p), anti‐phosphorylated histone H3 (1:200, Cell Signaling Technology, #9701), anti‐α‐SMA (1:4, DAKO, #U7033), anti‐active β‐catenin (1:200, Cell Signaling Technology, #19807), anti‐E‐cadherin (1:100, BD, #610182), anti‐CD31 (1:100, BD, #557355), anti‐CD68 (1:100, AbD Serotec, #MAC1957), anti‐Ly6G (1:100, Abcam, #ab25377), anti‐CD45 (1:150, BD, #550539), anti‐P‐Smad1/5 (1:50, Cell Signaling Technology, #9516), anti‐integrin α5 (1:100, Abcam, #ab150361), and anti‐integrin β1 (1:1,000, Abcam, #ab179471).

    Techniques: Co-Culture Assay, In Vitro, Recombinant, Activity Assay, Derivative Assay

    ANGPTL 2 does not directly activate β‐catenin signaling in epithelial cells in vitro Experimental protocol of organoid regeneration assay. Dissociated organoid cultures treated with vehicle or rANGPTL2. Scale bar = 200 μm. Organoid growth efficiency and size after passage treated with vehicle or rANGPTL2 ( n = 4). Data from vehicle were set at 1. Organoid cultures following 1‐Gy irradiation and treated with vehicle or rANGPTL2. Scale bar = 200 μm. Organoid growth efficiency and size after 1‐Gy irradiation and treated with vehicle or rANGPTL2 ( n = 12). Data from vehicle were set at 1. mRNA levels of indicated transcripts in colon and epithelium from wild‐type mice ( n = 4) based on qRT–PCR analysis. Intα5 , integrin α5 ; Intβ1 , integrin β1 . Integrin expression in HEK293, Caco‐2, and SW480 cells. Representative profiles obtained by FACS analysis using indicated anti‐integrin antibodies (black line traces) or control IgG (filled gray traces). Western blotting analysis of HEK293, Caco‐2, and SW480 cells treated with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. HSC70 serves as an internal control. Data information: Data are represented as means ± SEM. ns, not statistically significant. (D) Unpaired Student's t ‐test, (F) unpaired Student's t ‐test. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis

    doi: 10.15252/embj.201695690

    Figure Lengend Snippet: ANGPTL 2 does not directly activate β‐catenin signaling in epithelial cells in vitro Experimental protocol of organoid regeneration assay. Dissociated organoid cultures treated with vehicle or rANGPTL2. Scale bar = 200 μm. Organoid growth efficiency and size after passage treated with vehicle or rANGPTL2 ( n = 4). Data from vehicle were set at 1. Organoid cultures following 1‐Gy irradiation and treated with vehicle or rANGPTL2. Scale bar = 200 μm. Organoid growth efficiency and size after 1‐Gy irradiation and treated with vehicle or rANGPTL2 ( n = 12). Data from vehicle were set at 1. mRNA levels of indicated transcripts in colon and epithelium from wild‐type mice ( n = 4) based on qRT–PCR analysis. Intα5 , integrin α5 ; Intβ1 , integrin β1 . Integrin expression in HEK293, Caco‐2, and SW480 cells. Representative profiles obtained by FACS analysis using indicated anti‐integrin antibodies (black line traces) or control IgG (filled gray traces). Western blotting analysis of HEK293, Caco‐2, and SW480 cells treated with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. HSC70 serves as an internal control. Data information: Data are represented as means ± SEM. ns, not statistically significant. (D) Unpaired Student's t ‐test, (F) unpaired Student's t ‐test. Source data are available online for this figure.

    Article Snippet: Nuclei were stained with DAPI for 20 min. Antibodies used were as follows: anti‐Ki67 (1:800, Leica Microsystems, #NCL‐Ki67p), anti‐phosphorylated histone H3 (1:200, Cell Signaling Technology, #9701), anti‐α‐SMA (1:4, DAKO, #U7033), anti‐active β‐catenin (1:200, Cell Signaling Technology, #19807), anti‐E‐cadherin (1:100, BD, #610182), anti‐CD31 (1:100, BD, #557355), anti‐CD68 (1:100, AbD Serotec, #MAC1957), anti‐Ly6G (1:100, Abcam, #ab25377), anti‐CD45 (1:150, BD, #550539), anti‐P‐Smad1/5 (1:50, Cell Signaling Technology, #9516), anti‐integrin α5 (1:100, Abcam, #ab150361), and anti‐integrin β1 (1:1,000, Abcam, #ab179471).

    Techniques: In Vitro, Irradiation, Mouse Assay, Quantitative RT-PCR, Expressing, FACS, Western Blot, Recombinant

    The Angptl2 −/− mouse colon shows decreased β‐catenin signaling Representative images of H E (A)‐ and PAS (B)‐stained colon sections from wild‐type and Angptl2 −/− mice. Scale bar = 100 μm. Representative images of colon crypts as assessed by Ki67 (C) and pH3 (D) staining. Scale bar = 100 μm. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild‐type ( n = 4) and Angptl2 −/− ( n = 4) mice based on qRT–PCR analysis. Data are represented as means ± SEM. * P

    Journal: The EMBO Journal

    Article Title: ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis

    doi: 10.15252/embj.201695690

    Figure Lengend Snippet: The Angptl2 −/− mouse colon shows decreased β‐catenin signaling Representative images of H E (A)‐ and PAS (B)‐stained colon sections from wild‐type and Angptl2 −/− mice. Scale bar = 100 μm. Representative images of colon crypts as assessed by Ki67 (C) and pH3 (D) staining. Scale bar = 100 μm. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild‐type ( n = 4) and Angptl2 −/− ( n = 4) mice based on qRT–PCR analysis. Data are represented as means ± SEM. * P

    Article Snippet: Nuclei were stained with DAPI for 20 min. Antibodies used were as follows: anti‐Ki67 (1:800, Leica Microsystems, #NCL‐Ki67p), anti‐phosphorylated histone H3 (1:200, Cell Signaling Technology, #9701), anti‐α‐SMA (1:4, DAKO, #U7033), anti‐active β‐catenin (1:200, Cell Signaling Technology, #19807), anti‐E‐cadherin (1:100, BD, #610182), anti‐CD31 (1:100, BD, #557355), anti‐CD68 (1:100, AbD Serotec, #MAC1957), anti‐Ly6G (1:100, Abcam, #ab25377), anti‐CD45 (1:150, BD, #550539), anti‐P‐Smad1/5 (1:50, Cell Signaling Technology, #9516), anti‐integrin α5 (1:100, Abcam, #ab150361), and anti‐integrin β1 (1:1,000, Abcam, #ab179471).

    Techniques: Staining, Mouse Assay, Isolation, Quantitative RT-PCR

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    Beta1-integrin is degraded and locates to the perinuclear region. All treatments were performed with 4.3 μM LecB. (A) Cells were treated as indicated for different time periods and β1-integrin protein levels were determined by Western blot analysis and densitometric quantification using ImageJ. Protein levels were normalized to actin. Representative blots (above) and quantification data (below) are depicted. Values represent the mean of at least three independent experiments ± SEM. Asterisks indicate the statistical significance. (B) Cells were treated as indicated for 1 h and 5 h and analyzed by confocal fluorescence microscopy with immunostainings for β-catenin (green), β1-integrin (red) and counterstaining for DNA (DAPI, blue).

    Journal: Biochimica et Biophysica Acta

    Article Title: Pseudomonas aeruginosa lectin LecB inhibits tissue repair processes by triggering β-catenin degradation

    doi: 10.1016/j.bbamcr.2016.02.004

    Figure Lengend Snippet: Beta1-integrin is degraded and locates to the perinuclear region. All treatments were performed with 4.3 μM LecB. (A) Cells were treated as indicated for different time periods and β1-integrin protein levels were determined by Western blot analysis and densitometric quantification using ImageJ. Protein levels were normalized to actin. Representative blots (above) and quantification data (below) are depicted. Values represent the mean of at least three independent experiments ± SEM. Asterisks indicate the statistical significance. (B) Cells were treated as indicated for 1 h and 5 h and analyzed by confocal fluorescence microscopy with immunostainings for β-catenin (green), β1-integrin (red) and counterstaining for DNA (DAPI, blue).

    Article Snippet: Following antibodies were used for Western blots in this study: anti-β-catenin (Abcam, #ab32572), anti-β-actin (Sigma, #A5316), anti-pAkt (Ser473) (CST, #4060), anti-Akt (pan) (CST, #A4691), anti-pGSK-3β (Ser9) (CST, #5558), anti-GSK (pan) (CST, #9315), anti-β1-integrin (Millipore, #MAB2000), anti-p65 pSer536 (CST, #3033), anti-p65 pSer 468 (CST, #3039), anti-cyclin E2 (CST, #4132), anti-cyclin B1 (CST, #12,231), anti-Rb pan (CST, #9309), anti-Rb pSer795 (CST, #9301), anti-rabbit-HRP (CST, #7074), anti-mouse-HRP (CST, #7076).

    Techniques: Western Blot, Fluorescence, Microscopy

    PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Inhibition, shRNA

    PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Migration, Over Expression, Transfection, Plasmid Preparation, Transwell Invasion Assay, Microscopy

    PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: In Vitro, In Vivo, Kinase Assay, Glutathione S-Transferase Assay, Staining, Purification, Transfection, Incubation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Plasmid Preparation, Fluorescence, Expressing

    Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Over Expression, Transfection, Scratch Wound Assay Assay, Plasmid Preparation, Expressing

    PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Inhibition, shRNA

    PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Migration, Over Expression, Transfection, Plasmid Preparation, Transwell Invasion Assay, Microscopy

    PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: In Vitro, In Vivo, Kinase Assay, Glutathione S-Transferase Assay, Staining, Purification, Transfection, Incubation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Plasmid Preparation, Fluorescence, Expressing

    Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P

    Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).

    Techniques: Over Expression, Transfection, Scratch Wound Assay Assay, Plasmid Preparation, Expressing

    Expression levels of THBS2 in tissue samples of AAA patients and organ donors (CTL) quantified by Western Blot. ( A ) Bands obtained in Western blot analysis of THBS2 in AAA and CTL tissue samples (representative image of three samples of each group); ( B ) Bar graph of expression levels of THBS2 in tissue specimens of AAA ( n = 11) and CTL ( n = 7) group calculated as a relative expression using α-tubulin for normalization; ( C ) Correlation of log (THBS2/α-tubulin) levels and miRNA-195-5p (2 −∆Ct ) expression in tissue samples ( n = 18, CTL and AAA). *, p

    Journal: International Journal of Molecular Sciences

    Article Title: Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients

    doi: 10.3390/ijms21134600

    Figure Lengend Snippet: Expression levels of THBS2 in tissue samples of AAA patients and organ donors (CTL) quantified by Western Blot. ( A ) Bands obtained in Western blot analysis of THBS2 in AAA and CTL tissue samples (representative image of three samples of each group); ( B ) Bar graph of expression levels of THBS2 in tissue specimens of AAA ( n = 11) and CTL ( n = 7) group calculated as a relative expression using α-tubulin for normalization; ( C ) Correlation of log (THBS2/α-tubulin) levels and miRNA-195-5p (2 −∆Ct ) expression in tissue samples ( n = 18, CTL and AAA). *, p

    Article Snippet: Primary specific antibodies were ITGAV (Integrin α5), dilution 1:1000, LASP1 (LIM and SH3 domain protein 1), dilution 1:4000, and LAMB3 (Laminin 3β subunit), dilution 1:1000, rabbit polyclonal antibodies (Proteintech, Rosemont, IL, USA); COL11A1 (Collagen XI α1), dilution 1:500, and TSP-2 (THBS2), dilution 1:1000, rabbit polyclonal antibodies (GeneTex, Hsinchu City, Taiwan), and α-tubulin mouse monoclonal (Cell Signalling, Leiden, The Netherlands) as reference.

    Techniques: Expressing, Western Blot

    mTORC2 regulates stemness of GBM stem-like cells. ( a ) Representative immunoblot and coimmunoprecipitation demonstrated higher mTORC2 activity and formation as evidenced by increased mTOR Ser2481 phosphorylation and association with Rictor in CD133-positive stem-like cells derived from U87MG. APC-conjugated CD133 antibody was from Mylteni Biotech. ( b ) Immunoblot analysis showing enhanced mTOR Ser2481 and Gli2 FL in GBM stem-like cells compared with normal human glial stem cells. ( c ) Rictor was overexpressed in LN229 cells. Simultaneously, Gli2 was knocked down in Rictor-overexpressed LN229 cells and cultured in stem cell medium. Rictor-overexpressed cells showed enhanced neurosphere formation but were unable to form large colonies when Gli2 was knocked down. ( d ) Both the number of CD133-positive cells and their expression were higher in Rictor-overexpressed LN229 cells as revealed by flow cytometric analysis. ( e ) Representative western blots showed increased levels of Oct4, Sox2, Nanog, Integrin α 6 and Nestin in Rictor-overexpressed LN229 cells. Nestin antibody was from Biolegend. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: mTORC2 regulates hedgehog pathway activity by promoting stability to Gli2 protein and its nuclear translocation

    doi: 10.1038/cddis.2017.296

    Figure Lengend Snippet: mTORC2 regulates stemness of GBM stem-like cells. ( a ) Representative immunoblot and coimmunoprecipitation demonstrated higher mTORC2 activity and formation as evidenced by increased mTOR Ser2481 phosphorylation and association with Rictor in CD133-positive stem-like cells derived from U87MG. APC-conjugated CD133 antibody was from Mylteni Biotech. ( b ) Immunoblot analysis showing enhanced mTOR Ser2481 and Gli2 FL in GBM stem-like cells compared with normal human glial stem cells. ( c ) Rictor was overexpressed in LN229 cells. Simultaneously, Gli2 was knocked down in Rictor-overexpressed LN229 cells and cultured in stem cell medium. Rictor-overexpressed cells showed enhanced neurosphere formation but were unable to form large colonies when Gli2 was knocked down. ( d ) Both the number of CD133-positive cells and their expression were higher in Rictor-overexpressed LN229 cells as revealed by flow cytometric analysis. ( e ) Representative western blots showed increased levels of Oct4, Sox2, Nanog, Integrin α 6 and Nestin in Rictor-overexpressed LN229 cells. Nestin antibody was from Biolegend. Statistical significance compared with the control is indicated by * P

    Article Snippet: Antibodies for mTOR(2983), phospho-mTOR (Ser2481) (2974), Rictor(9476), GSK-3β (9315), β -Actin(4970), Gli1(3538S), Ptch1(2468S), Sufu(2522S), Shh(2207S), Snail(3879S), Slug(9585S), VEGF(2463S), Cyclin D1(2978S), Cyclin D2(3741S), Cyclin E(81045S), Oct4(2840), Sox2(3579), Nanog(4903), Integrin α 6(3750), HDAC3(2632S), rabbit IgG(3900S), HRP-conjugated anti-rabbit antibodies (7047S) and anti-mouse secondary antibodies (7076) were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activity Assay, Derivative Assay, Cell Culture, Expressing, Flow Cytometry, Western Blot

    mTORC2 regulates the expression of Gli1 and Gli2. U87MG cells were transfected with shRictor_1 and shRictor_2 shRNA ( a – c ) and LN229 cells were transfected with myc-tag wild-type Rictor (pRK-5/Rictor) for Rictor overexpression ( d – f ). After 36 h, cells were processed for western blotting, coimmunoprecipitation and real-time PCR analysis. RNA isolation kits were from Qiagen. ( a ) Representative immunoblot and coimmunoprecipitation of mTOR and Rictor confirmed reduced mTORC2 formation, as well as lower mTORC2 activity, as shown by decreased phosphorylation of mTOR at Ser2481 in U87MG cells after Rictor knockdown. ( b ) Real-time PCR analysis of Gli1, Gli2, Gli3 and Ptch1 mRNA expression in U87MG cells after mTORC2 disruption by shRictor_1 and shRictor_2 relative to that of untransfected cells. Values are normalized against 18S rRNA expression ( n =3 experiments). ( c ) Representative immunoblot showing reduced Gli1, Gli2 FL and Ptch1 protein levels and increased Gli3 Rep upon Rictor knockdown in U87MG cells. However, there was no apparent change in protein level of Sufu, Smo and Shh. ( d ) Representative immunoblots and coimmunoprecipitation experiments confirmed enhanced mTORC2 formation, as well as increased mTORC2 activity, as shown by higher mTOR Ser2481 phosphorylation in Rictor-overexpressed LN229 cells. ( e ) Real-time PCR analysis of Gli1, Gli2, Gli3 and Ptch1 mRNA expression in LN229 cells after mTORC2 activation relative to that of untransfected cells. Values are normalized against 18S rRNA expression ( n =3 experiments). ( f ) Representative immunoblots showing increased Gli1, Gli2 FL , Ptch1 and decreased Gli3 Rep protein levels upon enhanced mTORC2 activity in LN229 cells, whereas Sufu, Shh and Smo protein levels remain almost equivalent. ( g ) Dual luciferase assay was performed using Gli reporter construct (Qiagen) in U87MG and LN229 cells. According to the manufacturer’s protocol, U87MG cells were transfected with negative control, positive control, Gli reporter and Gli reporter with Rictor shRNA. Similarly, LN229 cells were transfected with negative control, positive control, Gli reporter and Gli reporter with pRK-5Rictor/Hh inhibitor (GANT61, 100 nM). Cells were processed for dual luciferase assay after 48 h using the Promega Dual Luciferase Kit according to the manufacturer’s instructions. Graphs show the mean±S.D. percentage of a relative luciferase unit after normalizing by comparing with control (incubated with vehicle only). Experiments were done in triplicate. Statistical significance compared with the control is indicated by * P

    Journal: Cell Death & Disease

    Article Title: mTORC2 regulates hedgehog pathway activity by promoting stability to Gli2 protein and its nuclear translocation

    doi: 10.1038/cddis.2017.296

    Figure Lengend Snippet: mTORC2 regulates the expression of Gli1 and Gli2. U87MG cells were transfected with shRictor_1 and shRictor_2 shRNA ( a – c ) and LN229 cells were transfected with myc-tag wild-type Rictor (pRK-5/Rictor) for Rictor overexpression ( d – f ). After 36 h, cells were processed for western blotting, coimmunoprecipitation and real-time PCR analysis. RNA isolation kits were from Qiagen. ( a ) Representative immunoblot and coimmunoprecipitation of mTOR and Rictor confirmed reduced mTORC2 formation, as well as lower mTORC2 activity, as shown by decreased phosphorylation of mTOR at Ser2481 in U87MG cells after Rictor knockdown. ( b ) Real-time PCR analysis of Gli1, Gli2, Gli3 and Ptch1 mRNA expression in U87MG cells after mTORC2 disruption by shRictor_1 and shRictor_2 relative to that of untransfected cells. Values are normalized against 18S rRNA expression ( n =3 experiments). ( c ) Representative immunoblot showing reduced Gli1, Gli2 FL and Ptch1 protein levels and increased Gli3 Rep upon Rictor knockdown in U87MG cells. However, there was no apparent change in protein level of Sufu, Smo and Shh. ( d ) Representative immunoblots and coimmunoprecipitation experiments confirmed enhanced mTORC2 formation, as well as increased mTORC2 activity, as shown by higher mTOR Ser2481 phosphorylation in Rictor-overexpressed LN229 cells. ( e ) Real-time PCR analysis of Gli1, Gli2, Gli3 and Ptch1 mRNA expression in LN229 cells after mTORC2 activation relative to that of untransfected cells. Values are normalized against 18S rRNA expression ( n =3 experiments). ( f ) Representative immunoblots showing increased Gli1, Gli2 FL , Ptch1 and decreased Gli3 Rep protein levels upon enhanced mTORC2 activity in LN229 cells, whereas Sufu, Shh and Smo protein levels remain almost equivalent. ( g ) Dual luciferase assay was performed using Gli reporter construct (Qiagen) in U87MG and LN229 cells. According to the manufacturer’s protocol, U87MG cells were transfected with negative control, positive control, Gli reporter and Gli reporter with Rictor shRNA. Similarly, LN229 cells were transfected with negative control, positive control, Gli reporter and Gli reporter with pRK-5Rictor/Hh inhibitor (GANT61, 100 nM). Cells were processed for dual luciferase assay after 48 h using the Promega Dual Luciferase Kit according to the manufacturer’s instructions. Graphs show the mean±S.D. percentage of a relative luciferase unit after normalizing by comparing with control (incubated with vehicle only). Experiments were done in triplicate. Statistical significance compared with the control is indicated by * P

    Article Snippet: Antibodies for mTOR(2983), phospho-mTOR (Ser2481) (2974), Rictor(9476), GSK-3β (9315), β -Actin(4970), Gli1(3538S), Ptch1(2468S), Sufu(2522S), Shh(2207S), Snail(3879S), Slug(9585S), VEGF(2463S), Cyclin D1(2978S), Cyclin D2(3741S), Cyclin E(81045S), Oct4(2840), Sox2(3579), Nanog(4903), Integrin α 6(3750), HDAC3(2632S), rabbit IgG(3900S), HRP-conjugated anti-rabbit antibodies (7047S) and anti-mouse secondary antibodies (7076) were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Transfection, shRNA, Over Expression, Western Blot, Real-time Polymerase Chain Reaction, Isolation, Activity Assay, Activation Assay, Luciferase, Construct, Negative Control, Positive Control, Incubation